The present invention relates in general to assays for detecting resistance of a retrovirus to reverse transcriptase inhibitor therapies and more specifically relates to a non-culture, polymerase chain reaction-based phenotypic assay for detecting antiviral drug-resistant reverse transcriptase activity in a sample from a patient infected with a retrovirus.
One of the most ravaging diseases of the late twentieth century has been AIDS (acquired immunodeficiency syndrome), brought on by HIV (human immunodeficiency virus) infection. Currently, there are no cures for this disease and minimally effective treatments. One of the problems that exists in the development of therapies for HIV infection is that the HIV virus rapidly develops resistance to a wide variety of chemotherapeutic agents. HIV, particularly human immunodeficiency virus type 1 (HIV-1), mutates over time to become resistant to many of the antiviral drugs administered for treatment. AIDS physicians need to know when the antiviral therapy being used to treat a individual patient is no longer effective so that the antiviral drug or drug combination can be modified, thereby minimizing viral replication and the onset of immunodeficiency symptoms.
Reverse transcriptase (RT) inhibitors such as zidovudine (ZDV, also referred to as azidothyimidine (AZT)), didanosine (ddI or dideoxyinosine), zalcitabine (ddC or dideoxycytosine), lamivudine (3TC), stavudine (d4T), and nevirapine (NVP) are nucleoside or non-nucleoside analogs currently approved for the treatment of HIV-1 infections. 3TC is known to have potent anti-HIV-1 activity and minimal toxicity, and is one of the most commonly used drugs in combination therapy as first-line treatment for HIV-1-infected patients. 3TC administered in combination with AZT provides greater and more sustained increases in CD4+ cell counts, and higher reductions in HIV-1 RNA viral load than continued AZT or 3TC monotherapy. 3TC in combination with AZT and protease inhibitors slows the progression of HIV-1 disease and reduces levels of HIV-1 RNA to less than 500 copies per milliliter for as long as one year in 90% of patients (Gulick et al., N. Engl. J. Med. 1997;337:734-739).
However, the use of reverse transcriptase inhibitors, such as 3TC, in both monotherapy or combination therapy has resulted in the emergence of drug-resistant variants of HIV-1 (Gulick et al., 1997). For a drug such as 3TC, the resistance is conferred by mutations in codon 184 of the HIV-1 reverse transcriptase gene, which replaces the wild type methionine residue (M; ATG) with a valine (V; GTG) via a transient substitution with an isoleucine (I; ATA). The presence of this M184V mutation has been associated with a greater than 500-fold resistance to 3TC and the partial loss of the anti-retroviral and clinical benefits of the drug. It is therefore important to monitor for drug resistance in individuals treated with reverse transcriptase inhibitors.
Phenotypic assays provide direct and definitive evidence of resistance to reverse transcriptase inhibitor drugs. However, presently available assays for the analysis of phenotypic resistance are based on virus culture and are therefore labor intensive and time consuming (two to five weeks), costly, and unsuitable for rapid clinical monitoring or surveillance of drug resistance (Kavlick et al., Antiviral Research 1995;28:133-146; Wainberg et al., AIDS 1995;9:351-357). In addition, these assays are fraught with biologic variabilities, including those related to viral isolation and tropism. Since tissue culture is highly selective for viral strains with in vitro growth advantages, these culture-based assay methods may not be representative of the total virus population present in vivo (Li et al., J. Virol. 1991;65:3973-3985].
In the absence of rapid phenotypic assays, genotypic tests are currently being used to provide indirect evidence of resistance. Genotypic testing monitors for the presence of mutations that are associated with resistance, such as the M184V mutation. Among these genotypic tests, primer-specific PCR, point mutation, and reverse hybridization assays are the most commonly used (Wainberg et al., 1995; Frenkel et al., J. Clin. Microbiol. 1995;33:342-347; Stuyver et al., Antimicrob. Agents Chemother. 1997;41:284-291). Unfortunately, clinical monitoring of reverse transcriptase inhibitor drug resistance by genotypic testing may not detect unrecognized mutations or potential synergistic or antagonistic effects of complex mutation patterns arising from combination therapy with different reverse transcriptase inhibitors. For example, the suppression of phenotypic resistance to AZT conferred by the M184V mutation clearly illustrates the effect that a combination of mutations may have in a given phenotype (Larder et al., Science 1995;269:696-699). Also, genotypic testing only detects resistance associated with known mutations (i.e., codon 184 for 3TC resistance).
U.S. Pat. No. 5,631,128 to Kozal describes polymerase chain reaction (PCR) assays for monitoring antiviral therapies in the treatment of AIDS. These genotypic assays use PCR to measure HIV-1 RNA copy number in plasma or to measure specific known HIV-1 RNA mutations, namely the mutation at codon 215 or codon 74 of the pol gene. The HIV-1 DNA copy number is an indication of the circulating HIV viral load. A decrease in HIV-1 RNA copy number correlates with successful antiretroviral therapy, whereas an increase in HIV-1 RNA copy number indicates disease progression, most likely caused by resistance to therapy. Therefore, the genotypic assays described in U.S. Pat. No. 5,631,128 detect only previously identified viral RNA mutations and are incapable of detecting phenotypic resistance caused by known or novel mutations, or the assays detect a rise in HIV-1 RNA copy number, which could be due to conditions other than resistance. An incorrect diagnosis of drug resistance followed by cessation of the antiviral therapy being administered could result in exacerbation of a disease that had been responding to therapy.
Therefore, there is a need for sensitive, rapid methods for the detection of HIV resistance to drug therapies in patients so that, if the virus becomes resistant to a particular drug or combination of drugs, the therapy can be modified, thereby keeping viral replication to a minimum and preventing or postponing the onset of AIDS.
An assay and kit for the detection of phenotypic resistance to a reverse transcriptase inhibitor drug in a biological sample is provided. Preferably, the biological sample is from a patient infected with a retrovirus. The assay is based on the direct analysis of the susceptibility of retroviral reverse transcriptase to inhibition by a reverse transcriptase inhibitor drug.
The enzymatic activity of the reverse transcriptase enzyme is determined by measuring the DNA product produced when an RNA template and a first complementary DNA primer from a suitable region of the encephalomyocarditis virus genome are incubated with a biological sample containing reverse transcriptase in the presence of the drug to which resistance is being determined. As a control, the enzymatic activity of the reverse transcriptase enzyme is also determined in the absence of the drug. The incubation mixture is reacted under conditions whereby the RNA template and the DNA primer will anneal and a DNA strand will be synthesized as an extension from the DNA primer if the reverse transcriptase in the sample is resistant to and not inhibited by the drug. The DNA product is amplified using a second complementary DNA primer from the encephalomyocarditis virus genome and suitable PCR reagents and conditions, and the amplified product detected in accordance with methods known to those skilled in the art. Detection of the amplified DNA indicates resistance to the drug employed in the assay. The difference in reverse transcriptase activity in the assays with and without drug verifies a finding of resistance and provides an indication as to the degree of resistance to the drug.
Preferably, the biological sample under investigation is a biological fluid, most preferably 0.5 xcexcl to 1.0 ml of blood plasma or serum Preferably, the RNA template consists of the ribonucleotide of SEQ ID NO:4; the first DNA primer consists of the oligonucleotide of SEQ ID NO:2; the second DNA primer consists of the oligonucleotide of SEQ ID NO:1; and PCR amplification is achieved by utilizing 30-40 cycles of heating the synthesized DNA and primer pair to 93 to 97xc2x0 C. for 30 to 90 seconds, at 53 to 57xc2x0 C. for 30 to 90 seconds, and at 70 to 74xc2x0 C. for 30 to 90 seconds. The amplified synthesized DNA is preferably detected by hybridization to an internal specific oligoprobe using an enzyme linked immunosorbent assay (ELISA), Southern blot hybridization methods, or similar methods.
Additionally provided is a kit for determining reverse transcriptase inhibitor drug resistance in a biological sample. The kit contains a suitable region of the encephalomyocarditis virus genome as an RNA template, a first complementary DNA primer for reverse transcriptase, and a second complementary DNA primer for amplification via the polymerase chain reaction, and the RT inhibitor or inhibitors under investigation, whereby each component is provided in separate containers or any combination of the components is provided in a single container. The kit may optionally contain the apparatus and one or more containers for obtaining and storing the sample prior to and during analysis and suitable buffers and other reagents to facilitate nucleic acid hybridization, synthesis, amplification and detection.
Therefore, it is an object of the present invention to provide sensitive methods for detecting drug resistance to reverse transcriptase inhibitor in a retrovirus-infected sample.
It is a further object of the present invention to provide a method for detecting drug resistance that is rapid, reliable, sensitive, and not labor intensive.
It is a further object of the present invention to provide a drug resistance assay that is phenotypic, not genotypic.
It is a further object of the present invention to provide an assay for drug resistance in which only a small amount of sample is needed for highly sensitive analysis.
It is a further object of the invention to provide an assay for the direct testing of biological body fluid samples such as serum, plasma, cerebrospinal fluid, saliva, semen and the like without extensive concentration, culturing or other processing techniques that would be required to increase the levels of reverse transcriptase in the sample under analysis.
It is a further object of the invention to provide an assay for drug resistance of a retrovirus that does not involve detection or amplification of the nucleic acid molecules of the retrovirus.
These and other objects, features, and advantages of the present method and kit will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.